Hypersensitivity Reactions In Mice Research Articles

Desloratadine ameliorates paclitaxel-induced peripheral neuropathy and hypersensitivity reactions in mice.

Paclitaxel (PTX) serves as a primary chemotherapy agent against diverse solid tumors including breast cancer, lung cancer, head and neck cancer and ovarian cancer, having severe adverse effects including PTX-induced peripheral neuropathy (PIPN) and hypersensitivity reactions (HSR). A recommended anti-allergic agent diphenhydramine (DIP) has been used to alleviate PTX-induced HSR. Desloratadine (DLT) is a third generation of histamine H1 receptor antagonist, but also acted as a selective antagonist of 5HTR2A. In this study we investigated whether DLT ameliorated PIPN-like symptoms in mice and the underlying mechanisms. PIPN was induced in male mice by injection of PTX (4 mg/kg, i.p.) every other day for 4 times. The mice exhibited 50% reduction in mechanical threshold, paw thermal response latency and paw cold response latency compared with control mice. PIPN mice were treated with DLT (10, 20 mg/kg, i.p.) 30 min before each PTX administration in the phase of establishing PIPN mice model and then administered daily for 4 weeks after the model wasestablished. We showed that DLT administration dose-dependently elevated the mechanical, thermal and cold pain thresholds in PIPN mice, whereas administration of DIP (10 mg/kg, i.p.) had no ameliorative effects on PIPN-like symptoms. We found that the expression of 5HTR2A was selectively elevated in the activated spinal astrocytes of PIPN mice. Spinal cord-specific 5HTR2A knockdown by intrathecal injection of AAV9-5Htr2a-shRNA significantly alleviated the mechanical hyperalgesia, thermal and cold hypersensitivity in PIPN mice, while administration of DLT (20 mg/kg) did not further ameliorate PIPN-like symptoms. We demonstrated that DLT administration alleviated dorsal root ganglion neuronal damage and suppressed sciatic nerve destruction, spinal neuron apoptosis and neuroinflammation in the spinal cord of PIPN mice. Furthermore, we revealed that DLT administration suppressed astrocytic neuroinflammation via the 5HTR2A/c-Fos/NLRP3 pathway and blocked astrocyte-neuron crosstalk by targeting 5HTR2A. We conclude that spinal 5HTR2A inhibition holds promise as a therapeutic approach for PIPN and we emphasize the potential of DLT as a dual-functional agent in ameliorating PTX-induced both PIPN and HSR in chemotherapy. In summary, we determined that spinal 5HTR2A was selectively activated in PIPN mice and DLT could ameliorate the PTX-induced both PIPN- and HSR-like pathologies in mice. DLT alleviated the damages of DRG neurons and sciatic nerves, while restrained spinal neuronal apoptosis and CGRP release in PIPN mice. The underlying mechanisms were intensively investigated by assay against the PIPN mice with 5HTR2A-specific knockdown in the spinal cord by injection of adeno-associated virus 9 (AAV9)-5Htr2a-shRNA. DLT inhibited astrocytic NLRP3 inflammasome activation-mediated spinal neuronal damage through 5HTR2A/c-FOS pathway. Our findings have supported that spinal 5HTR2A inhibition shows promise as a therapeutic strategy for PIPN and highlighted the potential advantage of DLT as a dual-functional agent in preventing against PTX-induced both PIPN and HSR effects in anticancer chemotherapy.

Antibodies against Poly(ethylene glycol) Activate Innate Immune Cells and Induce Hypersensitivity Reactions to PEGylated Nanomedicines.

Nanomedicines and macromolecular drugs can induce hypersensitivity reactions (HSRs) with symptoms ranging from flushing and breathing difficulties to hypothermia, hypotension, and death in the most severe cases. Because many normal individuals have pre-existing antibodies that bind to poly(ethylene glycol) (PEG), which is often present on the surface of nanomedicines and macromolecular drugs, we examined if and how anti-PEG antibodies induce HSRs to PEGylated liposomal doxorubicin (PLD). Anti-PEG IgG but not anti-PEG IgM induced symptoms of HSRs including hypothermia, altered lung function, and hypotension after PLD administration in C57BL/6 and nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Hypothermia was significantly reduced by blocking FcγRII/III, by depleting basophils, monocytes, neutrophils, or mast cells, and by inhibiting secretion of histamine and platelet-activating factor. Anti-PEG IgG also induced hypothermia in mice after administration of other PEGylated liposomes, nanoparticles, or proteins. Humanized anti-PEG IgG promoted binding of PEGylated nanoparticles to human immune cells and induced secretion of histamine from human basophils in the presence of PLD. Anti-PEG IgE could also induce hypersensitivity reactions in mice after administration of PLD. Our results demonstrate an important role for IgG antibodies in induction of HSRs to PEGylated nanomedicines through interaction with Fcγ receptors on innate immune cells and provide a deeper understanding of HSRs to PEGylated nanoparticles and macromolecular drugs that may facilitate development of safer nanomedicines.

Topical Application of Adenosine A2-Type Receptor Agonists Prevents Contact Hypersensitivity Reactions in Mice by Affecting Skin Dendritic Cells

Adenosine (Ado) produced by skin and skin migratory CD73+ dendritic cells is critically involved in tolerance tohaptens. We therefore investigated the use of Ado receptor agonists for the treatment of contact hypersensitivity reactions. A2A- 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino] ethyl]benzenepropanoic acid hydrochloride (CGS) and A2B- 2-[[6-Amino-3,5-dicyano-4-[4-[cyclopropylmethoxy]phenyl]-2-pyridinyl]thio]-acetamide (BAY) specific Ado receptor agonists were epicutaneously applied to the skin before sensitization and challenge with DNFB. Both agonists reduced ear swelling compared with solvent controls. This was accompanied by fewer activated T cells in the skin after the challenge and by higher numbers of T cells expressing anergic markers such as LAG-3, CD137, PD-1, CD272, and TIM-3 in the lymph nodes of CGS-treated groups. In ear tissue, Ado receptor agonist treatment reduced the production of proinflammatory cytokines and chemokines as well as the infiltration by neutrophils after sensitization. Moreover, reduced numbers of skin migratory dendritic cells producing less IL-12 and exhibiting lower expression of CD86 were recorded in lymph nodes after sensitization. In cocultures of skin migratory dendritic cells from CGS-treated mice with T cells, reduced proliferation of T cells and decreased secretion of proinflammatory cytokines compared with that of solvent controls were apparent. In conclusion, topical application of Ado receptor agonists to the skin prevents sensitization of T cells against haptens by reducing the migration and activation of skin migratory dendritic cells.

Penilloic acid is the chief culprit involved in non-IgE mediated, immediate penicillin-induced hypersensitivity reactions in mice.

Metabolites/impurities (MIs) of penicillin are normally considered to be the main substances inducing immediate hypersensitivity reactions in penicillin treatment. Our previous research found that penicillin can cause non-allergic hypersensitivity reactions (NAHRs) by directly triggering vascular hyperpermeability and exudative inflammation. However, the chief culprits and underlying mechanisms involved in penicillin-induced NAHRs have not yet been fully elucidated. In this study, we used a combination of approaches including a mouse non-allergic hypersensitivity reaction model, UPLC-MS/MS analyses of arachidonic acid metabolites (AAMs), immunoblotting technique, and molecular docking, etc to investigate the culprits involved in penicillin-induced hypersensitivity reactions. We found penilloic acid, one of the main MIs of penicillin, could trigger NAHRs via inducing increased vascular permeability, while the other MIs did no exhibit similar effect. Penilloic acid-induced reactions were not IgE-dependent. Significantly increased arachidonic acids and cascade metabolites in lungs, and activation of RhoA/ROCK signaling pathway in the ears and lungs of mice were noticed after once administration of penilloic acid. This study revealed that penilloic acid was the chief culprit involved in penicillin-induced immediate NAHRs in mice, which mainly associated with direct stimulation of vascular hyperpermeability and exudative inflammation. The activations of AAMs and RhoA/ROCK signaling pathway played important roles in these reactions.

Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

Chemotherapy-induced peripheral neuropathy (CIPN) and hypersensitivity reactions (HSRs) are among the most frequent and impairing side effects of the antineoplastic agent paclitaxel. Here, we demonstrated that paclitaxel can bind and activate complement component 5a receptor 1 (C5aR1) and that this binding is crucial in the etiology of paclitaxel-induced CIPN and anaphylaxis. Starting from our previous data demonstrating the role of interleukin (IL)-8 in paclitaxel-induced neuronal toxicity, we searched for proteins that activate IL-8 expression and, by using the Exscalate platform for molecular docking simulations, we predicted the high affinity of C5aR1 with paclitaxel. By in vitro studies, we confirmed the specific and competitive nature of the C5aR1-paclitaxel binding and found that it triggers intracellularly the NFkB/P38 pathway and c-Fos. In F11 neuronal cells and rat dorsal root ganglia, C5aR1 inhibition protected from paclitaxel-induced neuropathological effects, while in paclitaxel-treated mice, the absence (knock-out mice) or the inhibition of C5aR1 significantly ameliorated CIPN symptoms—in terms of cold and mechanical allodynia—and reduced the chronic pathological state in the paw. Finally, we found that C5aR1 inhibition can counteract paclitaxel-induced anaphylactic cytokine release in macrophages in vitro, as well as the onset of HSRs in mice. Altogether these data identified C5aR1 as a key mediator and a new potential pharmacological target for the prevention and treatment of CIPN and HSRs induced by paclitaxel.

Исследование общей и специфической токсичности рекомбинантного ангиогенина человека

Angiogenin exerted no toxic effects in acute toxicity tests, all animals successfully endured the maximally attainable dose 12 g/kg intragastrically. To assess the long-term/chronic toxicity, the gel-based composition containing 0.01 % of angiogenin was daily applied to the scarified skin areas of rats for 1 month. No significant changes or differences between the treated and control groups were detected by common biochemical, hematological and histological assays. No local irritating effects of 0.0025 % angiogenin gel were detected in appropriate tests using outbred chinchilla rabbits. Angiogenin did not cause neither sensibilization of guinea pigs in active cutaneous anaphylaxis test nor delayed hypersensitivity reaction in mice. No detectable immuno- and embryotoxicity of the preparation were observed. Angiogenin was proved to lack mutagenic and carcinogenic capabilities, as well as the capability of stimulating the tumor growth upon intratumoral administration. The tested preparation of angiogenin showed no toxic effects, including systemic, local irritating, sensibilizing and skin-resorptive ones, and, thus, recombinant human angiogenin is a safe pharmacological substance. 10.30906/2073-8099-2022-14-2-40-53

Obesity aggravates contact hypersensitivity reaction in mice.

Obesity is associated with chronic, low-grade inflammation in tissues and predisposes to various complications, including inflammatory skin diseases. However, the link between obesity and contact hypersensitivity (CHS) is not fully understood. We sought to determine the influence of obesity on T helper 1 (Th1)-mediated CHS. The activity/phenotype/cytokine profile of the immune cells was tested in vivo and in vitro. Using quantitative polymerase chain reaction (qPCR) and fecal microbiota transplantation (FMT), we tested the role of a high-fat diet (HFD)-induced gut microbiota (GM) dysbiosis in increasing the effects of CHS. Exacerbated CHS correlates with an increased inflammation-inducing GM in obese mice. We showed a proinflammatory milieu in the subcutaneous adipose tissue of obese mice, accompanied by proinflammatory CD4+ T cells and dendritic cells in skin draining lymph nodes and spleen. Obese interleukin (IL)-17A-/-B6 mice are protected from CHS aggravation, suggesting the importance of IL-17A in CHS aggravation in obesity. Obesity creates a milieu that induces more potent CHS-effector cells but does not have effects on already activated CHS-effector cells. IL-17A is essential for the pathogenesis of enhanced CHS during obesity. Our study provides novel knowledge about antigen-specific responses in obesity, which may help with the improvement of existing treatment and/or in designing novel treatment for obesity-associated skin disorders.

Immunity response of mice infected with laser irradiated protoscoleces of Echinococcus granulosus

The present study investigated the effect of the Neodymium- Doped Yttrium Aluminum Garnet (Nd: Y3AL5G12) Laser eradiation 1000 mw, against infection with secondary hydatid disease in BALB/c mice by inoculating the animals with protoscoleces of Echinococcus granulosus exposed to laser irradiation, for various periods 40,60,120 and 240 minutes, with viability of 70%,57%,50% and 40%, respectively, contrasted to the control set (mice inoculated with unexposed protoscoleces) along three months, depending on the evaluation of the total count of WBCs and assessment of acquired cell- mediated immunity, demonstrated by delayed type hypersensitivity test (DTH). The results showed considerable increase (p<0.01) in the total WBCs counting, in mice injected with irradiated protoscoleces, up to 13962 cell/cm3, in comparison to the control group 5420 cell/cm3, after 120 minutes, two months post infection. Moreover, there was considerable excess (p<0.01) in the foot pad thickness in treated mice, 2.94 mm after 120 minute (24hour post antigen injection), two months post infection, 2.86mm after 120 minutes (24 hours post antigen injection), 1 month post infection and 2.64 mm, 120 minutes (24 hours post antigen injection), three months post infection, compared with the control group 1.18mm, 1.80mm and 1.72mm, respectively. Results revealed that irradiation of Echinococcus granulosus protoscoleces with laser had a significant influence on the production of WBCs and stimulation of delayed-type hypersensitivity reaction in mice.

Characterization of a TNFR2-Selective Agonistic TNF-α Mutant and Its Derivatives as an Optimal Regulatory T Cell Expander.

Regulatory T cells (Tregs) are a subpopulation of lymphocytes that play a role in suppressing and regulating immune responses. Recently, it was suggested that controlling the functions and activities of Tregs might be applicable to the treatment of human diseases such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease. TNF receptor type 2 (TNFR2) is a target molecule that modulates Treg functions. In this study, we investigated the role of TNFR2 signaling in the differentiation and activation of mouse Tregs. We previously reported the generation of a TNFR2-selective agonist TNF mutant, termed R2agoTNF, by using our unique cytokine modification method based on phage display. R2agoTNF activates cell signaling via mouse TNFR2. In this study, we evaluated the efficacy of R2agoTNF for the proliferation and activation of Tregs in mice. R2agoTNF expanded and activated mouse CD4+CD25+ Tregs ex vivo. The structural optimization of R2agoTNF by internal cross-linking or IgG-Fc fusion selectively and effectively enhanced Treg expansion in vivo. Furthermore, the IgG-Fc fusion protein suppressed skin-contact hypersensitivity reactions in mice. TNFR2 agonists are expected to be new Treg expanders.

347 Regulated expression of CD73 by subsets of dendritic cells in skin is crucial for modulating contact hypersensitivity reactions in mice

The ecto-5'-nucleotidase CD73 converts extracellular adenosine monophosphate, which is a product of ATP degradation by CD39, to adenosine (ADO). Because ADO has well established anti-inflammatory effects, expression of CD73 by different types of skin cells may be crucial for the outcome of contact hypersensitivity (CHS) reactions. At first we analysed expression of CD39 and CD73, respectively, by different subsets of skin dendritic cells (DCs) and found ubiquitous expression of CD39 on all defined DC subsets. In contrast, CD73 was expressed by only 3%-8% of CD207+ Langerhans cells (LC) and other dermal DCs, respectively. Similar expression patterns were observed in skin migrating (sm) DCs in the lymph node under steady state conditions. However, application of the hapten TNCB induced surface-expression of CD73 in all cells, resulting in 20%-30% CD73+ DC and LC, respectively. This increased expression of CD73 was persistent, as it was also detectable in smDC 24h-48h after sensitization. Next, CD73 deficient (CD73KO) mice were subjected to a classical TNCB-induced CHS protocol and we found increased ear swelling reactions as compared to wild type mice, indicating rather down-modulatory functions of CD73 in CHS reactions. To accurately assess the tolerogenic functions of CD73 expression by smDC in CHS reactions, we established a tolerance model whereby application of DNTB renders animals tolerant to subsequent sensitization with DNFB. Here we show that both, the tolerogen DNTB as well as the sensitizer DNFB induced migration of similar DC subsets from skin to lymph nodes. But after DNTB application substantially higher expression levels of CD73 was observed in particular in CD11b+ as well as in CD207+CD103+ smDC. Of note, CD73KO animals were resistant to tolerization by DNTB. Thus, these data indicate a substantial role of CD73+ skin DC in regulating tolerance to haptens.

Expression of CD73 slows down migration of skin dendritic cells, affecting the sensitization phase of contact hypersensitivity reactions in mice

BackgroundApplication of haptens to the skin induces release of immune stimulatory ATP into the extracellular space. This “danger” signal can be converted to immunosuppressive adenosine (ADO) by the action of the ectonucleotidases CD39 and CD73, expressed by skin and immune cells. Thus, the expression and regulation of CD73 by skin derived cells may have crucial influence on the outcome of contact hypersensitivity (CHS) reactions. ObjectiveTo investigate the role of CD73 expression during 2,4,6-trinitrochlorobenzene (TNCB) induced CHS reactions. MethodsWild type (wt) and CD73 deficient mice were subjected to TNCB induced CHS. In the different mouse strains the resulting ear swelling reaction was recorded along with a detailed phenotypic analysis of the skin migrating subsets of dendritic cells (DC). ResultsIn CD73 deficient animals the motility of DC was higher as compared to wt animals and in particular after sensitization we found increased migration of Langerin+ DC from skin to draining lymph nodes (LN). In the TNCB model this led to a stronger sensitization as indicated by increased frequency of interferon-γ producing T cells in the LN and an increased ear thickness after challenge. ConclusionCD73 derived ADO production slows down migration of Langerin+ DC from skin to LN. This may be a crucial mechanism to avoid over boarding immune reactions against haptens.

The toxicity of 3-monochloro-1,2-propanediol (+) to activated T cells in mice

ABSTRACT3-monochloro-1,2-propanediol (3-MCPD) is a member of a group of chemicals known as chloropropanols that has been detected in a wide range of foods. In the present study, we found that its α-chlorohydrin structure (3-MCPD(+)) has toxic effects on activated T cells in mice. Specifically, the data showed that 1, 2, and 4 mM 3-MCPD(+) markedly inhibited ConA-induced T cell proliferation and Th1/Th2 cytokine production. Furthermore, Western blotting was used to study the mechanism of action of 3-MCPD(+). The data revealed that 3-MCPD(+) inhibited the activation of the Ca2+/CaM/I-κB-NF-κB and Ca2+/CaM/CaN/NFAT signaling pathways. In vivo, 3-MCPD(+) treatment significantly inhibited 2,4-dinitrofluorobenzene-induced delayed-type hypersensitivity reactions in mice. These observations indicated that 3-MCPD(+) is toxic to activated mouse T cells in vitro and in vivo.

Involvement of Histamine and RhoA/ROCK in Penicillin Immediate Hypersensitivity Reactions.

The mechanism of penicillin immediate hypersensitivity reactions has not been completely elucidated. These reactions are generally considered to be mediated by IgE, but penicillin-specific IgE could not be detected in most cases. This study demonstrated that penicillin was able to cause vascular hyperpermeability in a mouse model mimicking clinical symptoms of penicillin immediate hypersensitivity reactions. The first exposure to penicillin also induced immediate edema and exudative reactions in ears and lungs of mice in a dose-dependent manner. Vasodilation was noted in microvessels in ears. These reactions were unlikely to be immune-mediated reactions, because no penicillin-specific IgE was produced. Furthermore, penicillin treatment directly elicited rapid histamine release. Penicillin also led to F-actin reorganization in human umbilical vein endothelial cells and increased the permeability of the endothelial monolayer. Activation of the RhoA/ROCK signaling pathway was observed in ears and lungs of mice and in endothelial cells after treatment with penicillin. Both an anti-histamine agent and a ROCK inhibitor attenuated penicillin immediate hypersensitivity reactions in mice. This study presents a novel mechanism of penicillin immediate hypersensitivity reactions and suggests a potential preventive approach against these reactions.

The Major Soybean Allergen Gly m Bd 28K Induces Hypersensitivity Reactions in Mice Sensitized to Cow’s Milk Proteins

Reactions to soy have been reported in a proportion of patients with IgE-mediated cow's milk allergy (CMA). In this work, we analyzed if Gly m Bd 28K/P28, one of the major soybean allergens, is a cross-reactive allergen with cow milk proteins (CMP). We showed that P28 was recognized by IgE sera from CMA patients and activated human peripheral basophils degranulation. Moreover, IgE sera of mice exclusively sensitized to CMP recognized P28. Splenocytes from sensitized animals secreted IL-5 and IL-13 when incubated with CMP or soy proteins, but only IL-13 when treated with P28. In addition, a skin test was strongly positive for CMP and weakly positive for P28. Remarkably, milk-sensitized mice showed hypersensitivity symptoms following sublingual challenge with P28 or CMP. With the use of bioinformatics' tools seven putative cross-reactive epitopes were identified. In conclusion, using in vitro and in vivo tests we demonstrated that P28 is a novel cross-reactive allergen with CMP.

Synthesis and biological evaluation of novel orally available 1-phenyl-6-aminouracils containing dimethyldihydrobenzofuranol structure for the treatment of allergic skin diseases.

We have designed and efficiently synthesized novel 1-phenyl-6-aminouracils by replacing the chroman moiety in CX-659S, a nonsteroidal dermatologic candidate, with dimethyldihydrobenzofuranol to cancel CX-659S asymmetric center. Medicinal chemistry effort culminated in the discovery of 13d bearing a 3-methyl group at the 1-phenyl group as a promising compound. Compound 13d, having good in vitro ADME profile and moderate oral bioavailability in mice, showed potent anti-inflammatory activity against hapten-induced contact hypersensitivity reaction in mice following topical and oral administration. The effects of 13d were equipotent to that of tacrolimus or prednisolone. In addition, compound 13d, having potent hydroxyl radical-scavenging activity, showed more potent suppressive effect on substance P-induced pruritus in mice than oxatomide.

Immunosuppressive activity of alpinetin on activation and cytokines secretion of murine T lymphocytes

Alpinetin, a flavonoid compound extracted from the seeds of Alpinia katsumadai Hayata, has been known to possess antibacterial, anti-inflammatory and other important therapeutic activities. In the current study, we investigated alpinetin for its immunosuppressive effect on activation and cytokines secretion of murine T lymphocytes. The data showed that alpinetin markedly suppressed ConA-induced murine splenocyte proliferation, Th1/Th2 cytokines production, CD4+ T-cell populations and ratio of CD4+/CD8+. This inspired us to further study the effects of alpinetin in vivo. The results showed that administration of alpinetin suppressed T-cell-mediated delayed-type hypersensitivity reaction in mice. In addition, we studied signal transduction pathways about T-cell activation on puried murine T lymphocytes by Western-blot assay. The data revealed that alpinetin could shock the activation of NF-κB, NFAT2 signal transduction pathways. These observations indicated that alpinetin have potential effects in downregulating the immune system and might be developed as a useful immunosuppressive agent in treating undesired immune responses.

A Critical Role for Thymic Stromal Lymphopoietin in Nickel-Induced Allergy in Mice

Ni is the most frequent cause of contact allergy induced by metals. However, the underlying mechanism of this induction is unknown. Our previous research demonstrates that activation of dendritic cells (DCs) through p38MAPK/MKK6 is required for Ni-induced allergy in mice. In the current study, we investigated the cellular and molecular mechanisms underlying Ni-induced allergy using a mouse model that involves injecting Ni into the ear, with or without Freund's incomplete or complete adjuvants. Nickel had greater potential to cause allergic reactions compared with palladium and gold. Among the proteins expressed at higher levels in mice with Ni-induced allergy, we focused on thymic stromal lymphopoietin (TSLP), which is produced in abundance by keratinocytes. We detected increased expression of the TSLP receptor (TSLPR) in DCs from cervical lymph nodes of mice with Ni-induced allergy, suggesting that DCs in ear tissues were activated through TSLPR signaling induced by keratinocyte-derived TSLP. Furthermore, delayed-type hypersensitivity reactions in mice with Ni-induced allergy were decreased significantly by injection of a Tslp-short interfering RNA along with atelocollagen in the ear skin. These results suggest that Ni allergy may be triggered by a TSLP/TSLPR-mediated interaction between epithelial and immune cells.

Suppressive effects of fisetin on mice T lymphocytes in vitro and in vivo

BackgroundMost of the immunosuppressive drugs have satisfactory therapeutic effects on organ transplantation and autoimmune disease. However, their clinical application is limited by side effects. Therefore, new and safe immunosuppressive drugs against acute and chronic rejections are eagerly awaited. Fisetin, a flavonoid present in various types of vegetables and fruits, has few side effects and low level of toxicity, which would be a desirable clinical feature. In the present study, we investigated the immunosuppressive effects and underlying mechanisms of fisetin against T-cell activation in vitro and in vivo. MethodsWe measured the effect of fisetin on T-lymphocyte proliferation, T-cell subsets, cell cycle progression, cytokine production, and nuclear factor activation in vitro, as well as its influence on T cell–mediated delayed-type hypersensitivity reaction in vivo. ResultsIn vitro, the results showed that fisetin significantly suppressed mouse splenocytes proliferation, Th1 and Th2 cytokine production, cell cycle and the ratio of CD4+/CD8+ T cells. Furthermore, fisetin exerts an immunosuppressive effect in mouse T lymphocytes through the suppression of nuclear factor kappa B activation and nuclear factor of activated T cells signaling in a dose-dependent manner. In vivo, fisetin treatment also significantly inhibited the dinitrofluorobenzene-induced delayed-type hypersensitivity reactions in mice. ConclusionsFisetin had strong immunosuppressive activity in vitro and in vivo, suggesting a potential role for fisetin as an immunosuppressive agent.

Pharmacologic Inhibition of IκB Kinase Activates Immediate Hypersensitivity Reactions in Mice

Pharmacologic inhibitors of IκB kinase (IKK), especially IKK-β, have been developed to treat inflammatory diseases. However, their interactions with components of the NF-κB pathways are not fully known in allergic diseases. To examine whether IKK is involved in immediate hypersensitivity reactions and to determine whether counterregulatory mechanisms in the NF-κB activation system were active, we examined the role played by IKK components on mast cell degranulation using a murine ocular immediate hypersensitivity reaction model. Pharmacologic inhibition of IKK in mice caused paradoxical aggravation of the mast cell-mediated immediate hypersensitivity reaction and up-regulation in the expression of inflammatory cytokines. Downstream analyses showed that B-cell deficiency or treatment by IL-1 receptor antagonist corrected the aberrant activation of tissue-resident mast cells, which would indicate contribution by activated B cells. Analyses of co-cultures of tissue-resident mast cells showed the contribution of activated B cells to activation of mast cells and secretion of inflammatory cytokines. Aberrant activation of the NF-κB promoter in isolated B cells was induced exclusively by IKK-β inhibition and was negated by ablating IKK-α. Aggravated mast cell degranulation by pharmacologic IKK inhibition in the murine immediate hypersensitivity reaction was corrected by B-cell-targeted inhibition of IKK-α. Thus, IKK-β limits B-cell-mediated mast cell activation and inflammatory cytokine induction in immediate hypersensitivity by counterbalancing the activity of IKK-α.

DX5+CD4+T cells modulate CD4+T‐cell response via inhibition of IL‐12 production by DCs

DX5(+) CD4(+) T cells have been shown to dampen collagen-induced arthritis and delayed-type hypersensitivity reactions in mice. These cells are also potent modulators of T-helper cell responses through direct effects on CD4(+) T cells in an IL-4 dependent manner. To further characterize this T-cell population, we studied their effect on DCs and the potential consequences on T-cell activation. Here, we show that mouse DX5(+) CD4(+) T cells modulate DCs by robustly inhibiting IL-12 production. This modulation is IL-10 dependent and does not require cell contact. Furthermore, DX5(+) CD4(+) T cells modulate the surface phenotype of LPS-matured DCs. DCs modulated by DX5(+) CD4(+) T-cell supernatant express high levels of the co-inhibitor molecules PDL-1 and PDL-2. OVA-specific CD4(+) T cells primed with DCs exposed to DX5(+) CD4(+) T-cell supernatant produce less IFN-γ than CD4(+) T cells primed by DCs exposed to either medium or DX5(-) CD4(+) T-cell supernatant. The addition of IL-12 to the co-culture with DX5(+) DCs restores IFN-γ production. When IL-10 present in the DX5(+) CD4(+) T-cell supernatant is blocked, DCs re-establish their ability to produce IL-12 and to efficiently prime CD4(+) T cells. These data show that DX5(+) CD4(+) T cells can indirectly affect the outcome of the T-cell response by inducing DCs that have poor Th1 stimulatory function.